CLINICAL OBSERVATIONS, INTERVENTIONS, AND THERAPEUTIC TRIALS
Flow cytometric disease monitoring in multiple myeloma: the relationship
between normal and neoplastic plasma cells predicts outcome after transplantation
Andy C. Rawstron, Faith E. Davies, Ranjit DasGupta, A. John Ashcroft, Russell Patmore, Mark T. Drayson,
Roger G. Owen, Andrew S. Jack, J. Anthony Child, and Gareth J. Morgan
Conventional monitoring strategies for
myeloma are not sufficiently sensitive to
identify patients likely to benefit from
further therapy immediately after transplantation. We have used a sensitive flow
cytometry assay that quantitates normal
and neoplastic plasma cells to monitor
the bone marrow of 45 patients undergoing high-dose chemotherapy. Neoplastic
plasma cells were detectable at 3 months
after transplantation in 42% of patients.
Once detected, neoplastic cell levels increased steadily until clinical progression: these patients had a significantly
shorter progression-free survival (PFS)
(median, 20 months) than those with no
detectable disease (median, longer than
35 months; P ⴝ .003). Neoplastic plasma
cells were detectable in 27% (9 of 33) of
immunofixation-negative complete-remission patients. These patients had a significantly shorter PFS than immunofixationnegative patients with no detectable
neoplastic plasma cells (P ⴝ .04). Normal
plasma cells were present in 89% of patients immediately after transplantation,
but were not sustained in most cases.
Patients with only normal phenotype
plasma cells present at 3 months after
transplantation and also at second assessment had a low risk of disease progression. Patients with neoplastic plasma
cells present at 3 months after transplantation, or with only normal plasma cells
present at first assessment and only neoplastic plasma cells at second assessment, had a significantly higher risk of
early disease progression (P < .0001) with
a 5-year survival of 54% for the high-risk
group, compared with 100% in the lowrisk group (P ⴝ .036). Analysis of normal
and neoplastic plasma cell levels is more
sensitive than immunofixation and can
identify which patients may benefit from
additional treatment strategies at an early
stage after transplantation. (Blood. 2002;
100:3095-3100)
© 2002 by The American Society of Hematology
Introduction
Many patients who receive high-dose therapy (HDT) for
myeloma achieve a complete remission by conventional criteria,
with a minority achieving a molecular remission. However, with
current therapy, all patients eventually relapse as a consequence
of residual disease. To develop effective maintenance strategies,
aimed at prolonging the residual disease states, it is important to
be able to monitor the behavior of residual neoplastic plasma
cells. In addition to monitoring the malignant cells, it has also been
suggested that recovery of normal immunoglobulin levels may be
associated with improved outcome.1 Monitoring the recovery of
normal plasma cells may therefore offer an additional approach to
predicting the outcome of autologous transplantation.
Current approaches to measurement of residual disease
levels are based on morphological assessment of bone marrow
biopsies, analysis of the paraprotein levels, or polymerase chain
reaction (PCR) analysis of the immunoglobulin heavy chain
variable-diversity-joining (VDJ) region. Complete remission
(CR) is currently defined as the absence of the original
monoclonal paraprotein in serum and urine by immunofixation
as well as fewer than 5% plasma cells in the bone marrow.2
Using these criteria, a number of studies have shown that
patients who achieve a complete remission have an improved
progression-free, and possibly also overall, survival compared
with partial responders or nonresponders.3-5 However, the
difference in survival is insufficient to justify using remission
status defined by conventional criteria as a means for adjusting
treatment. PCR strategies using primers specific to the neoplastic VDJ region result in sensitivities of up to 1 in 106 cells.
However, such approaches are not quantitative and are laborintensive, and only 60% to 70% of patients have an amplifiable
VDJ region.6 Therefore, these strategies are difficult to apply in
a clinical setting, and it is not clear whether they improve the
prediction of outcome after transplantation.7-9
An optimal assay for monitoring residual disease would be
robust and universally applicable, and could quantitate low levels
of neoplastic plasma cells. We have developed a flow cytometric
technique for identifying plasma cells with a sensitivity of 0.01%.
The assay can distinguish neoplastic plasma cells from their normal
counterparts on the basis of their CD19 and CD56 expression, even
if both cell types are present within the same sample.10,11 We have
applied this technique to bone marrow aspirates from a series of
patients undergoing autologous transplantation in order to determine whether the levels of malignant and normal plasma cells
predict outcome after high-dose therapy.
From the Academic Unit of Haematology and Oncology, University of Leeds,
United Kingdom; Department of Haematology, Hull Royal Infirmary, United
Kingdom; and Department of Immunology, University of Birmingham, United
Kingdom.
Reprints: Andy C. Rawstron, HMDS, Academic Unit of Haematology and
Oncology, Algernon Firth Bldg, University of Leeds, Leeds LS1 3EX, United
Kingdom; e-mail: [email protected].
Submitted December 21, 2001; accepted May 21, 2002. Prepublished online
as Blood First Edition Paper, June 7, 2002; DOI 10.1182/blood-2001-12-0297.
Supported by The Leukaemia Research Fund, United Kingdom; and Yorkshire
Cancer Research, United Kingdom.
BLOOD, 1 NOVEMBER 2002 䡠 VOLUME 100, NUMBER 9
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2002 by The American Society of Hematology
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BLOOD, 1 NOVEMBER 2002 䡠 VOLUME 100, NUMBER 9
Patients and methods
Patients
Forty-five patients were analyzed in this study: 24 male, 21 female. Median
age at presentation was 55 years (range, 41-65 years); ␤2 microglobulin
(␤2m) levels were lower than 345 nM (lower than 4 mg/L) in 49%, 346 to
690 nM (4 to 8 mg/L) in 36%; and greater than 690 nM (greater than 8
mg/L) in 18% of patients. The majority (39 of 45) were treated in the
Medical Research Council Myeloma VII trial high-dose arm. All patients
were treated with continuous intravenous adriamycin and vincristine over
4 days with pulsed intravenous corticosteroids and cyclophosphamide
(C-VAMP) repeated every 3 weeks to maximal response. Only patients who
achieved at least a partial response to C-VAMP proceeded to high-dose
therapy, which consisted of melphalan at 200 mg/m2 and high-dose
methylprednisolone with autologous stem cell support. Response was
monitored by means of serum electrophoresis and immunofixation at
3-month intervals. Complete response required fewer than 5% plasma cells
on bone marrow aspirate and biopsy and a negative serum electrophoresis
and immunofixation.2 Bone marrow aspirate samples were obtained at 3
months after transplantation, and then at intervals of 3 to 6 months
thereafter. Samples were analyzed prospectively.
Flow cytometry
Leukocytes were prepared by incubation with a 10-fold excess of ammonium chloride (8.6 g/L in distilled H20) for 5 minutes, and washed twice in
FACSFlow (BD Biosciences, Oxford, United Kingdom) containing 0.3%
bovine serum albumin (BSA) (Sigma-Aldrich, Dorset, United Kingdom).
Then, 1 ⫻ 106 leukocytes were stained with 10 ␮L volumes of each
pretitrated antibody per test for 20 minutes at 4°C, washed twice, and
acquired by means of a Becton Dickinson FACSort with CELLQuest v3.1
software (BD Biosciences). Cells were incubated with CD45 fluorescein
isothiocyanate (FITC) (inhouse); CD38–phycoerythrin/cyanin 5 (PE/Cy5)
(inhouse); and either CD3-PE (inhouse), CD138-PE (Serotec, Oxford,
United Kingdom), CD19-PE (inhouse), or CD56-PE (BD Biosciences,
Oxford, United Kingdom). Between 50 000 and 500 000 total cells were
analyzed in each test.
The gating strategy is optimized to exclude contaminating events, particularly B progenitors, which are common in samples from patients immediately
after transplantation, as well as apoptotic cells and cellular debris. Analysis of
CD38 versus CD138 expression (Figure 1Ai) provides the best separation of
plasma cells from other leukocytes, but is also subject to contamination, with
cells binding antibodies nonspecifically. This can be detected on the CD38 versus
CD45 plot (Figure 1Aiii) to the right of the plasma cell population. Thus, an
initial region (R1) is set around cells expressing a high level of CD38 and CD138
(Figure 1Ai), and a second region (R2) set on the light scatter of gated
CD38⫹CD138⫹ cells (Figure 1Aii). A third region (R3) was set around the cells
satisfying both R1 and R2 for CD38 and CD45 expression (Figure 1Aiii).
Regions R2 and R3 are then optimized until events falling within both of these
regions are all CD138⫹ and CD3⫺.
Horizontal quadrant markers are set according to the CD3 control for analysis
of CD138 and CD19 expression. CD56 expression is weak on normal plasma
cells, and the marker is set higher than control (at 100 as standard) as this provides
a better discrimination between normal and neoplastic cells. The expression of
CD19-PE and CD56-PE is then used to distinguish between normal and
neoplastic plasma cells. The former are consistently CD19⫹CD56dim whereas the
latter are CD19⫺ or CD19⫹CD56⫹.10,12 The level of CD19 expression is broad
on normal plasma cells, and up to 10% may be CD19⫺ compared with control.
Therefore, samples were classified as containing neoplastic plasma cells only if
more than 10% had an abnormal phenotype. A minimum of 50 events that
satisfied the gating strategy (Figure 1) were required for identification of a
neoplastic or normal plasma cell population. Up to 500 000 events were acquired,
allowing a maximum sensitivity of detection of 0.01% in all cases.
Dilute nonrepresentative aspirate samples are a significant problem in all
minimal residual disease studies. This assay allows the identification of such
samples in most cases. Those containing fewer than 0.01% normal or neoplastic
Figure 1. Flow cytometric detection of neoplastic plasma cells. (A) The gating
strategy used to detect plasma cells, designed to exclude the majority of contaminating events (particularly B-progenitor cells, apoptotic cells, and cellular debris)
common in posttreatment samples, described in detail in “Patients and methods.”
(B) Representative plots from patients at 3 months after transplantation. The top row
shows a patient whose bone marrow sample contains only normal phenotype
(CD19⫹CD56dim) plasma cells. The middle row shows a sample with only neoplastic
phenotype (CD19⫺ or CD19⫹CD56⫹) plasma cells. The bottom row shows a sample
containing mostly normal plasma cells with a detectable neoplastic population that
represents 15% of total plasma cells.
plasma cells were always unrepresentative of the trephine biopsy appearance and
were not included in the study. In this series of patients, unsuitable aspirates were
received in approximately 6% (3 of 48) of cases. Marrow aspirate samples
containing both neoplastic and normal plasma cells were always representative,
as normal CD138⫹ plasma cells are found only in bone marrow, not in peripheral
blood.10 All patients with disease on the trephine biopsy had neoplastic plasma
cells detectable by flow cytometry, although the degree of infiltration was
underestimated in a minority of cases.
Fluorescent immunoglobulin heavy chain gene PCR analysis
using consensus primers
High molecular weight DNA was obtained from separated leukocytes by
proteinase K digestion, phenol/chloroform extraction, and cold ethanol
precipitation. DNA was amplified with a 5⬘ FITC-labeled primer to a
consensus region of the J heavy chain (JH) gene, and a primer to a
consensus framework 3 (Fr3) region, or a mixture of primers to
consensus framework 1 (Fr1) regions on each of the 6 VH gene families,
as reported previously.6 Electrophoresis and analysis was performed by
means of an Applied Biosystems (Foster City, CA) automated DNA
sequencer. Electrophoretograms were produced from the fluorescence
intensity data, representing the size and relative amount of each PCR
product as a peak on a histogram. This PCR assay will detect neoplastic
plasma cells at the level of 1 in 105 leukocytes if no other B cells are
present. If B cells are present, which is the case in most patient samples,
the assay will identify a population that represents more than 2% of total
BLOOD, 1 NOVEMBER 2002 䡠 VOLUME 100, NUMBER 9
amplifiable B cells, equating to a sensitivity of 1 in 103 to 1 in 104 total
leukocytes.13
Results
Sensitivity of flow cytometry assay: comparison with
consensus-primer immunoglobulin heavy chain–PCR
We have previously demonstrated that fluorescent consensus-primer
immunoglobulin heavy chain (IgH)–PCR has a comparable sensitivity
for the detection of residual disease to immunofixation.3 To determine
whether flow cytometric assessment would be more applicable, we
compared the flow assay with consensus-primer IgH-PCR analysis.
Twenty-five patients had amplifiable DNA from presentation marrow
samples, of which 16 of 25 (64%) had an amplifiable IgH rearrangement. This is consistent with previous studies demonstrating an amplifiable rearrangement in up to 80% of patients.6,14 The flow assay detected
neoplastic plasma cells at presentation in all patients included in this
study, and we have previously demonstrated in a series of more than 500
patients that the assay will detect neoplastic plasma cells in more than
98% of cases.15
Thirty-three follow-up samples were available from patients
with an amplifiable IgH rearrangement. Neoplastic plasma cells
were detected by flow cytometry in all PCR-positive samples
(n ⫽ 10) and also in 16 of 23 PCR-negative samples. In PCRnegative samples, neoplastic plasma cell levels were below 0.2% of
total leukocytes (median, 0.06%), consistent with the limits of
sensitivity of the PCR assay in patient samples.13 The results
indicate that flow cytometric analysis is applicable to a greater
proportion of patients than IgH-PCR in general, and also has a
greater sensitivity for detection of residual neoplastic cells than
consensus-primer IgH-PCR.
Comparison of conventional monitoring with flow
cytometric analysis
In this group of patients, 22% (10 of 45) achieved an immunofixation-negative complete remission, with the remaining 78% (35 of
45) achieving a partial response to induction therapy with CVAMP. High-dose melphalan increased the complete remission
rate to 73% (33 of 45), with 27% (12 of 45) remaining immunofixation positive. Neoplastic plasma cells were detectable at 3 months
after transplantation in 27% (9 of 33) of the complete remission
patients, and in 92% (11 of 12) of partial remission patients. To
determine the reproducibility of the technique, 34 representative
samples were reanalyzed retrospectively in a blinded fashion.
Differences were noted in only 2 of 34 samples; in 1 case the
difference was due to operator error on prospective analysis. In the
other case, neoplastic plasma cells were present at a level close to
the limit of detection of the flow assay, and the sample was
prospectively reported as showing a normal plasma cell profile, but
having evidence of residual disease on retrospective analysis. The
clinical features suggest that the retrospective analysis was more
accurate, as the patient remained immunofixation positive. However, to avoid potential bias, the results reported in this paper are
from prospective analysis. This retrospective analysis demonstrates
that the assay has good reproducibility, but reanalysis of borderline
samples using larger numbers of cells may be beneficial in
future studies.
For patients achieving a complete remission after transplantation, the median time to achieve a negative immunofixation was 2.9
PHENOTYPING PREDICTS TRANSPLANT OUTCOME IN MYELOMA
3097
months after transplantation, with 12% (4 of 33) taking longer than
6 months to show undetectable levels of paraprotein. To assess
whether neoplastic plasma cell levels showed the same kinetics,
sequential samples were assessed in a cohort of 12 patients with
neoplastic plasma cells detectable at 3 months after transplantation.
A median of 2 further samples were analyzed (range, 1-7) with a
median follow-up of 15 months from transplantation. In 11 of 12
patients, the levels of neoplastic plasma cells increased steadily; for
1 patient, the level of neoplastic plasma cells was stable (ie, within
0.05% of previous samples) for 17 months and then increased until
clinical relapse occurred at 38 months. Thus, once neoplastic
plasma cells are detected, the levels increase until overt clinical
progression.
These data suggest that analysis of neoplastic plasma cells is
more sensitive than immunofixation in the majority of cases, and
can be performed at a single time point, as once neoplastic cells are
detected they do not decrease in level.
Use of the flow cytometric assay improves prediction
of outcome compared with standard criteria
In this series of patients, attainment of an immunofixation-negative
complete remission was associated with improved progression-free
survival, but this only became apparent at 20 months after
transplantation (Figure 2).
Patients with detectable paraprotein by immunofixation had a
median progression-free survival of 21.5 months (95% confidence
interval, 20-23 months), whereas only 12 of 33 immunofixationnegative patients have progressed with a median 30-month follow
up (P ⫽ .002, log-rank test). Overall survival at 5 years was 77%
for the complete responders compared with 52% for the partial
responders, but this did not reach statistical significance (P ⫽ .16,
log-rank test).
When the presence or absence of neoplastic plasma cells was
used to define response, a similar pattern was seen, although there
was slightly better separation between the 2 arms in the first year
after transplantation compared with conventional monitoring
(Figure 3).
Figure 2. Prolonged progression-free survival in patients attaining a complete
remission. Kaplan-Meier analysis of progression-free and overall survival, comparing patients achieving an immunofixation-negative complete remission against those
achieving a partial remission only. Survival is shown from time of transplantation.
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RAWSTRON et al
Neoplastic plasma cells were detectable in the bone marrow of
42% of patients (19 of 45) at 3 months after autologous transplantation. Patients with detectable neoplastic plasma cells had a median
progression-free survival of 20 months (95% confidence interval,
18-23 montha), whereas only 9 of 26 in the group of patients with
no neoplastic plasma cells have progressed with a median 34.5
months follow-up (P ⫽ .0034, log-rank test). Survival at 5 years
was 64% for the group with neoplastic plasma cells compared with
76% for those with no detectable neoplastic cells, although again
this did not reach significance (P ⫽ .28, log-rank test).
Neoplastic plasma cells were detectable in 9 of 33 (27%) of the
immunofixation-negative (IF⫺) patients, and these patients had a
median progression-free survival (PFS) of 20 months. This was
significantly poorer than the 24 IF⫺ patients with no detectable
neoplastic plasma cells: only 7 of 24 (29%) have progressed, with a
median follow-up of 34.5 months (P ⫽ .04, log-rank test). There
was no difference in overall survival. Immunofixation provided no
additional information with respect to progression-free survival for
patients with detectable neoplastic plasma cells present. Of 19
patients with neoplastic plasma cells at 3 months after transplantation, 9 became immunofixation-negative and had a median PFS of
20 months, whereas 10 remained immunofixation positive and had
a median PFS of 22 months (P ⫽ .6, log-rank test).
Univariate and multivariate analysis of outcome was performed
for a range of criteria shown in Table 1. The detection of
paraprotein by immunofixation was not significant in multivariate
analysis; however, the detection of neoplastic plasma cells at 3
months remained significant. This demonstrates the increased
sensitivity of the flow cytometric assay for detection of residual
disease. In patients with detectable neoplastic plasma cells, approximately half become immunofixation negative but show an outcome
identical to patients who remain immunofixation positive. Presentation ␤2m level was the only other variable to remain significant on
multivariate analysis, demonstrating that the presence of residual
disease is an independent prognostic factor. Thus, the flow assay
can discriminate a group of patients who are at risk of relapsing
Figure 3. Prediction of early relapse from the presence of neoplastic plasma
cells at 3 months after transplantation. The presence of neoplastic plasma cells at
3 months after transplantation predicts early relapse. Kaplan-Meier analysis of
progression-free and overall survival, comparing patients with detectable neoplastic
plasma cells at 3 months after transplantation against those with only normal plasma
cells present. Survival is shown from time of transplantation.
Table 1. Only presentation ␤2m levels and detection of neoplastic plasma cells
at 3 months after transplantation are significant predictors of outcome in
multivariate analysis
Univariate
Multivariate
Age at transplantation, older than 55 years
Factor
0.426
NT
Sex, male
0.078
NT
Presentation creatinine, greater than 130 ␮M
0.267
NT
Presentation hemoglobin, less than 12 g/dL
0.021
0.442
Presentation ␤2m, greater than 4.0 mg/L
0.016
0.050
Immunofixation-negative after transplantation
0.002
0.786
Neoplastic plasma cells at 3 months after transplantation
0.003
0.014
Univariate analysis (log-rank test) of factors that may affect progression-free
survival, and multivariate Cox-regression analysis of factors significant in
univariate analysis.
NT indicates not tested.
early despite achieving an immunofixation-negative complete
remission, independent of presentation ␤2m levels.
Normal plasma cells are present in the bone marrow of most
patients at 3 months after transplantation. If this level is
sustained for 6 months, patients have a significantly
better prognosis
At 3 months after transplantation, normal plasma cells were
detectable in 89% (40 of 45) of patients, and normal CD19⫹
B-lymphocytes were present in all patients. Previous studies have
suggested that the recovery of normal immunoglobulin is a
powerful prognostic factor, but the proportion of patients recovering normal levels is much lower. Analysis of sequential samples in
25 patients demonstrated that 16 of 25 patients had normal plasma
cells at second assessment (6 to 12 months after transplantation),
and all 16 of these patients recovered normal immunoglobulin
levels. These patients had a much better prognosis: only 2 of 16
have progressed at 40 and 49 months after transplantation,
respectively, with a median follow-up of 39 months. However, the
median time to recovery of normal immunoglobulin levels was 6
months, and ranged from 3 to 15 months. Therefore, recovery of
normal immunoglobulin levels is not a suitable parameter for
identifying patients requiring further upfront therapy. Of the 9
patients who had only neoplastic plasma cells at second assessment, all had continued immuneparesis. The outcome of the
latter group was similar to the poor outcome of patients with
detectable neoplastic plasma cells at 3 months, showing a
median progression-free survival of 16 months from transplantation (range, 7-39 months).
The data suggest that it is possible to identify 2 groups of
patients with very different outcomes, on the basis of the detection
of normal and neoplastic plasma cells at first and second assessment after transplantation. In a cohort of 35 patients, we defined a
high-risk group (n ⫽ 23) as those who have neoplastic plasma cells
present at 3 months after transplantation or who have only normal
plasma cells present at first assessment and only neoplastic plasma
cells at second assessment. The low-risk group had only normal
phenotype plasma cells present at 3 months after transplantation
and also had normal plasma cells present at second assessment
(n ⫽ 12). Figure 4 demonstrates a highly significant difference in
progression-free survival between these groups (P ⬍ .0001). In
addition, there is also a significant difference in overall survival
(P ⫽ .036), with a 5-year survival of 100% for the low-risk group,
compared with 54% in the high-risk group.
BLOOD, 1 NOVEMBER 2002 䡠 VOLUME 100, NUMBER 9
Figure 4. Prediction of survival by levels of normal and neoplastic plasma cells
immediately after transplantation. Levels of normal and neoplastic plasma cells
immediately after transplantation provide a powerful prediction of both progressionfree and overall survival. Kaplan-Meier analyses of progression-free and overall
survival for 2 groups of patients according to levels of neoplastic and normal
plasma cells are shown. Patients who have only normal cells after transplantation
and who sustain this recovery have a significantly improved progression-free and
overall survival. Those who have neoplastic plasma cells present after transplantation, or who recover normal plasma cells by 3 months after transplantation but
who have no normal plasma cells present at 6 to 12 months, have poor
progression-free and overall survival. Survival is shown from time of
transplantation.
Discussion
In this study, we have assessed the clinical relevance of minimal
disease monitoring in patients with multiple myeloma after
high-dose chemotherapy using conventional criteria, as well as
flow cytometric and PCR approaches. Flow cytometric analysis
has not been widely used as a method for residual disease
analysis in multiple myeloma, but has been previously shown to
be extremely effective in chronic lymphocytic leukemia.13 It has
been demonstrated that a unique neoplastic plasma cell phenotype is identifiable in more than 98% of patients, and that the
technique can be extremely sensitive.15 We have demonstrated
in this study that a relatively simple flow cytometric technique
can identify a group of patients with particularly good prognosis, and a second group with a much poorer progression-free and
overall survival.
More recently identified neoplastic markers may allow further
improvement of flow cytometric disease monitoring in myeloma.16
However, during this study, we have identified several factors that are
essential for residual disease assessment. Factors that affect any residual
disease assay are the use of good quality “first-pull” marrow aspirate and
the analysis of sufficient leukocytes (preferably 100 000 to 500 000).
Particularly relevant to flow cytometric analysis is the exclusion of
B-progenitor cells, which have a phenotype similar to that of normal
plasma cells (CD38⫹⫹CD19⫹CD56⫺). These are best excluded by their
lack of CD138 expression.
Numerous PCR strategies have been applied for residual
disease monitoring in myeloma, with some showing an improved outcome for those achieving an minimal residual
PHENOTYPING PREDICTS TRANSPLANT OUTCOME IN MYELOMA
3099
disease–negative (MRD-negative) status,8 and some showing no
difference.9 A major drawback to PCR analysis is the relatively
low number of patients with an amplifiable IgH rearrangement
using consensus primers, as a result of a high degree of somatic
hypermutation in the neoplastic cells.6 Analysis using consensus
primer PCR is relatively insensitive and provides no additional
information to what is provided by immunofixation.3 Allelespecific oligonucleotide (ASO)–PCR approaches are more sensitive, but also more labor-intensive and cannot differentiate
between myeloma plasma cells and clonally related B cells. This
is critical since some investigators have detected B cells
clonally related to the myeloma plasma cells after transplantation, yet their presence does not predict early relapse, possibly
because these cells may not be proliferative.17,18 This may
explain why most patients remain ASO-PCR positive after
autologous transplantation and why monitoring does not always
predict outcome in this setting.7
As in previous studies, we have demonstrated that patients
achieving a complete remission have an improved progression-free
survival compared with partial remission patients.4 However, this
difference becomes apparent only after prolonged follow-up, and
many CR patients show early relapse. Furthermore, it may take up
to 9 months after transplantation for immunofixation to become
negative. Therefore, conventional criteria cannot be used to
identify patients who might benefit from additional therapy in the
early stages after high-dose therapy.
Flow cytometric analysis of normal and neoplastic plasma
cells provides a much more powerful prediction of outcome in
this cohort and can be assessed at fixed time points as the result
does not depend on variable immunoglobulin half-life. It has
been suggested that neoplastic plasma cell levels might remain
stable over time in some patients after transplantation.19 However, in this study, neoplastic plasma cell levels always increased with time until disease progression, and their identification at an early stage predicts a poor outcome. Patients who had
only normal plasma cells at first assessment but only neoplastic
plasma cells at second assessment also had a poor outcome. This
distinct group of patients who respond well initially but relapse
quickly has previously been identified in studies of conventional
chemotherapy.20 Patients with only normal plasma cells at first
assessment and who maintain normal plasma cells at second
assessment are nearly all in remission with a median follow-up
from transplantation of approximately 3 years. These patients
also show recovery of normal immunoglobulin levels that have
previously been identified as a good predictor of outcome.1 It
seems probable that this group of patients will not benefit from
additional therapy immediately after transplantation but should
be monitored regularly for residual disease on maintenance
therapy. In contrast, patients with detectable neoplastic plasma
cells at 3 or 6 months after transplantation should be considered
for further treatment, such as further high-dose therapy, lowintensity conditioning allogeneic transplantation, or experimental therapeutic strategies.
Acknowledgment
The majority of patients investigated in this study were treated in
the Medical Research Council Myeloma VII trial (Adult Leukaemia Working Party Chairman Prof A. K. Burnett).
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